CN1272879A - Attenuated Japanese encephalitis virus adapted to vero cell and Japanese encephalitis vaccine - Google Patents

Attenuated Japanese encephalitis virus adapted to vero cell and Japanese encephalitis vaccine Download PDF

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CN1272879A
CN1272879A CN98809797A CN98809797A CN1272879A CN 1272879 A CN1272879 A CN 1272879A CN 98809797 A CN98809797 A CN 98809797A CN 98809797 A CN98809797 A CN 98809797A CN 1272879 A CN1272879 A CN 1272879A
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vaccine
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japanese encephalitis
vero cell
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CN1142271C (en
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金炫洙
刘王敦
金寿玉
李星熙
文祥帆
洪璿杓
申营喆
郑用周
K·H·埃克尔斯
B·英尼斯
J·R·普特纳克
L·N·宾尼
A·K·斯里瓦斯塔瓦
D·R·杜波依斯
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Reed Institute For Military Research
First Sugar Refining Corp
CJ Corp
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Abstract

The present invention relates to an attenuated Japanese encephalitis virus adapted to Vero cell by passages on Vero cell and a Japanese encephalitis vaccine comprising said attenuated virus.

Description

A kind of japanese encephalitis virus and Japanese encephalitis vaccine that adapts to the attenuation of African green monkey kidney cell
Background of invention
Invention field
The present invention relates to a kind of japanese encephalitis virus of the attenuation by adapting to the Vero cell at African green monkey kidney (Vero) passage and the Japanese encephalitis vaccine that comprises described attenuated virus.
Description of the Prior Art
Japanese encephalitis (JE) is that the area, Asia is to the very important a kind of arboviruses disease of being carried by mosquito of public health.It is reported, case and 10000 above people's death more than 35000 are arranged in the Asia every year, but official report has but been underestimated real case load (Okuno, T. " world's healthy state " (World Health Stat Q) 3:120-31,1978 undoubtedly; Umenei, people such as T., Bull World Health Org.63:625-31,1985).The performance of disease is fever and headache symptom, aseptic meningitis or encephalitis, and has the survivor of half to have persistent neuroscience and psychosis sequela (Burke, people such as D.S. " arboviruses: epidemiology and ecology " 3:63-92,1988 approximately; Monath, T.P. virusology 763-814,1990).
JE (Japanese encephalitis) virus is a kind of in the flavivirus in 66, and what mainly be that carrier carries tunicary positive has an adopted single strand RNA virus (Chambers, people such as T.J., Ann.Rev.Microbiol.44:649-88,1990).On form, flavivirus is spherical in shape, and diameter is about 40nm, forms by being centered around the outer double-layer of lipoid of nucleocapsid, and this nucleocapsid contains and glutelin (C) compound 11kb genome (Rice, people such as C.M., Science, 229:726-33,1985).Surface on the film is outstanding to be made up of glycosylated coating (E) and film (M) albumen.Preceding M glycoprotein in born of the same parents in the nascent virion fragments into the M albumen of finding in the virion of sophisticated extracellular.Important physical is active relevant with 53kd E albumen, and these activity comprise hemagglutination, virus neutralization, virion assembling, film merges and virus combines (Koshini, people such as E., Virol.188:714-20,1992) with cell receptor.
Existing three-type-person uses JE vaccine (Tsai, people such as T., vaccine, 671-713,1993).The JE vaccine of the deactivation that wherein only produces in mouse brain can be buied in the world.A manufacturer, Osaka university microbial diseases WARF (Biken) have produced the deactivation JE vaccine that great majority are sold in the world; This virus secured permission in the U.S. in 1992, and by Connaught Laboratories, Inc. is with JE-VAX TMTrade(brand)name sell.Deactivation that makes in former generation hamster kidney (PHK) cell and the attenuation JE vaccine of living are only sold in China.
The JE vaccine of the deactivation that produces in mouse brain secured permission in Japan in 1954.Owing to it produces by injecting young mouse brain, therefore produce very effort, and relate to the possibility that causes sick of vaccine related neural side effect.Although purity and effectiveness (Oye, A. vaccine inoculation theory and practice, 69-82,1975 that a series of improvement that production method is done have improved vaccine; Oya, A.Acta PediatrJpn.30:175-84,1988; Takaku, K.Biken J.11:25-39,1968), but up to date also report have the local reaction of certain frequency and slight systemic reaction (Hoke, people such as C.H., New Engl J Med.319:608-14,1988; Poland, people such as J.D., J InfectDis.161:878-82,1990; Sanchez, people such as J.L., Lancet 335:972-73,1990).Have 20% vaccine to produce locally to touch a tender spot in the injection site, rubescent and/or swelling.It is reported have the vaccine of 10-30% to produce slight systemic symptom, mainly be headache, low fever, myalgia, do not accommodate the stomach symptom.From 1989, the obvious new untoward reaction of having reported comprises urticaria, angioedema, respiratory distress, erythema multiforme, erythema nodosum (erythema nosodum) and serious neurological disease, these mainly are to be subjected to find among the visitor of vaccine inoculation (Anderson in Australia state, Europe and North America, M.M. wait the people, Lancet.337:1044,1991; Ruff, people such as T.A., Lancet 228:881-2,1991).In addition, 1992 and nineteen ninety-five, Ohtaki has reported 7 children through JE vaccine inoculation future trouble acute disseminated encephalomyelitis (ADEM), and changes (Ohtaki, people such as E., Pediatr Neutrol.8:137-9,1992 with magnetic resonance image (MRI) (MRI); Ohtaki, people such as E., J Neurol Neruosurg Psychiatry59:316-7,1995).Be also noted that with the Rabies Vaccine inoculation that contains animal brain and can cause serious neurological complication (Plotkin, people such as S.A., vaccine, 661-2,1994).For those reasons, WHO has paid the utmost attention to exploitation JE vaccine.
Recently, verified, the deactivation of China and the attenuation JE vaccine of living are that effectively it has brought out the antibody of the neutralization virus of high titre, and physical protection (Tsai, people such as T., vaccine, 671-713,1993) is provided.Yet the PHK cell for preparing Chinese vaccine is not used to produce virus vaccines by world health organization (WHO) approval, is not used for human by developed country's permission yet.Main drawback with former generation hamster cell production vaccine is the uncertainty of vaccine quality.Even adopt the hamster that does not have specific pathogen, animal still can be by inadvertent contamination, and this is a problem in the production of vaccine.Sometimes, this class infection energy is not detected for a long time.For these criticisms, need do further controlled research to vaccine safety, so that it is used widely is credible.Another shortcoming of producing vaccine from primary cell is that viral yield rate is low, the cost height, and can not mass production.
In view of the foregoing, need a kind of in standard clone such as African green monkey kidney cell or people's diploid cells (these cells by approval as people's vaccine stroma) in generation and lower-cost new JE vaccine.The Vero cell be derived from the monkey kidney through transforming but do not cause the cell of tumour.Vero clone is all more favourable than other any standard cell lines, because the easier adaptation mass cell cultivation of Vero cell, and as a kind of cell transformed, it has unlimited life.
Have been found that JE virus can grow in the Vero cell cultures.In JE vaccine field, done considerable effort and come to produce vaccine, thereby can carry out the cell cultures of large volume from standard cell lines.Yet, virus specificity analysis (comprising genetic stability), also must not satisfy the demand of people's vaccine by make required yield of vaccine commercialization and method from the Vero cell culture.Because these are true and be difficult to the knowledge that other virus culture obtains is used for JE virus, prior art is not successfully developed stable and JE virus vaccines that immunogenicity is high in heredity from continuous cell line.All these researchs all fail to draw a kind of new immunogenic production process that satisfies standard described in this background so far.
The present invention proposes a kind of in passage cell (Vero cell), the growth and method that propagation JE virus is used to produce vaccine, in mouse brain or primary cell line, produce the problem of JE virus before this method has overcome.The present invention has also determined to cultivate the method for JE virus and a kind of downstream processes of low cost production vaccine.
In addition, the present invention has determined original commercialization JE vaccine is done improvement on the following method.
1. security: virus of the present invention does not obtain toxicity by the Vero cell culture, has reduced the danger of producing, and provides extra security (except the strictness control security that virus inactivating method provided) for the recipient.Business-like JE vaccine in the past never provided this advantage.
2. the supply in safer production matrix increases: produce JE vaccine of the present invention at bovine serum in the presence of not, obtained the production of high yield and cheap and scalable scale, these are that original commercialization JE vaccine institute is irrealizable.
3. reactionogenicity still less (reactogenicity): in JE vaccine of the present invention, do not mix gelatine stabiliser, thereby reduced danger as the vaccine reaction of existing vaccine (people such as Saakaguchi M., vaccine, 68-69,1998).In addition, removed the unwanted ox of mixing in the existing JE vaccine composition of deriving effectively.In a word, former JE vaccine never provided this security of the present invention.
4. the effectiveness of Ti Gaoing: not the existing and effective purifying of successful, the additive that is used to produce of Vero cell large scale production, these make and can at first use effective adjuvant when preparation JE vaccines.Also being applicable to other vaccine although use adjuvant in vaccine formulation, is very difficult with this knowledge use in the JE vaccine, because there is not a kind of existing JE vaccine can guarantee the production that it is safe.
In a word, also do not produce a kind of new vaccine so far and can satisfy the business-like above-mentioned advantage of JE vaccine.
Therefore, an object of the present invention is to provide a kind of JE vaccine that safely and effectively, in the standard cell lines substrate, produces, to improve its acceptability in many countries.Another object of the present invention provides a kind of effective ways, is used to the vaccine of producing highly purified stable vaccine and preparing high immunogenicity with a small amount of antigen.
Summary of the invention
On the one hand, the invention provides a kind of japanese encephalitis virus of the attenuation that adapts to the Vero cell by on the Vero cell, going down to posterity.The japanese encephalitis virus of attenuation of the present invention is called CJ50003 in the text, according to Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure, be deposited in Korea S microorganism culturing center (Seoul on April 20th, 1998, Korea), can obtain this viral subculture from this preservation mechanism according to preserving number KCCM-10125.
On the other hand, the invention provides a kind of Japanese encephalitis vaccine, it contains the japanese encephalitis virus of the attenuation that adapts to Vero cell by going down to posterity on the Vero cell.
The accompanying drawing summary
Referring to behind following description, claims and the accompanying drawing, just can understand these and further feature, aspect and advantage of the present invention better, these accompanying drawings are as follows:
Fig. 1 has shown JE virus SA14-14-2 (PDK) going down to posterity and adapt in the Vero cell.With behind the SA 14-14-2 strain (PDK) of the 0.1moi inoculation Vero cell monolayer the 5th day, results first-generation virus.On the Vero cell monolayer, carry out the plaque test, measure the titre of JE virus.Use first-generation virus as parent material subsequently, carry out continuous virus infection and titration, went down to posterity for 30 generations according to embodiment 1 is described.
Fig. 2 has shown every other day rolling in the 3rd day to the 17th day after the infection and has repeatedly gathered in the crops JE virus CJ50003 in the bottle.Infection multiplicity vero cells infection individual layer with each cell 0.01 plaque forming unit (pfu).Make virus in 35 ℃ of absorption 2 hours, use the PBS washed cell then three times, add 100 milliliters of serum-free EMEM, 35 ℃ of cultivations.After infection the 3rd day to the 17th day, replaced culture supernatant with fresh serum-free EMEM every 48 hours.Carry out the viral infection titration of cutting by on the Vero cell monolayer, measuring plaque.
Fig. 3 has shown with SDS-PAGE and Western trace and has analyzed through the JE of the super centrifugal purification of saccharose gradient virus CJ50003.60 milliliters of spissated culture supernatant are added 40 milliliters of 15-60% saccharose gradients, and 12 ℃, 22000rpm are centrifugal 18 hours in 45 Ti rotary heads.Collect 2 ml samples from the test tube bottom, and carry out the SDS-PAGE of 4-20% gradient, the albumen of differentiating is transferred on the nitrocellulose filter.Develop the color by Xylene Brilliant Cyanine G (group A) or Silver Nitrate (group B) dyeing, antigen develops the color by monoclonal antibody (group C) reaction with responding property of JE virus E protein.Swimming lane 1 is prestained protein standard substance (Novex Seeblue TM), represent 250,98,64,50,36,30,16 and the molecular weight of 6kDa from the top; Swimming lane 2-10 is super centrifugal afterwards from the fraction No.3-11 of bottom; E, envelope protein; C, glutelin; M, membranin.
Fig. 4 has shown the formalin-inactivated kinetics of the JE virus CJ50003 of purifying.At 4 ℃ or 22 ℃ of JE virus prepared products with 0.018% formalin-inactivated purifying.By showing directly that on the Vero cell monolayer plaque comes the residual infection venereal disease poison of titration institute's sample thief during the fixed time.In addition, the test of increasing as described below is to detect low-level virus.The flask (in duplicate) that contains the Vero cell monolayer with the sample inoculation of inactivation of virus time point.35 ℃ of absorption add cell and 35 ℃ of cultivations after 2 hours again.After infection, added cell in the 7th day and the 14th day again.The results substratum also forms plaque, to detect infectious virus.The inactivation time point that has shown two different experiments among the figure: 4 ℃ (Filled Rectangle), 22 ℃ (solid circles).Parallelly carry out hot deactivation (not having formaldehyde) contrast (4 ℃ are expressed as hollow rectangle, and 22 ℃ are expressed as hollow circle).
Detailed Description Of The Invention
The present invention relates to a kind of JE Strain that has for the preparation of the desired character of JE vaccine. Described virus is one Plant the virus of attenuation, can breed in continuous cell line, this continuous cell line is that the WHO approval is given birth to as people's vaccine Produce with the Vero cell at the bottom of the cell based. Therefore, estimate that described virus can be used for preparing than safer the going out of existing vaccine Live with the JE vaccine of living.
The invention provides a kind of vaccine that satisfies present needs. By not being higher than 35 ℃ of lower continuous passages, make JE Virus adapts to the Vero cell. Continuous passage causes virus titer to be increased to and is higher than 10 in the Vero cell7The pfu/ milliliter Culture supernatant, and reduced the incubation time that shows the virus titer peak value. The present invention relates to a kind of Multiple harvests side The development of method, the method do not need serum as additive, produce the output yield height of virus, and this is for low cost The described vaccine of large-scale production is the character of viable commercial. According to the present invention, the Multiple harvests when virus is cultivated Method causes cytopathic effect (CPE) degree of infected cell to reduce. Because the minimizing of CPE level, this The residual cells that bright JE vaccine the contains minute quantity composition of deriving. In addition, estimate that JE vaccine of the present invention will provide Than the immunogenicity of existing JE vaccine enhancing and the disease-resistant protectiveness of raising. The JE vaccine of purifying of the present invention is with existing The major advantage that has vaccine to compare is, satisfied the development of vaccine for man from the virus of the Vero cell purification cultivated Need.
The invention still further relates to the method for the described vaccine of preparation.This method can provide the described vaccine of high yield, high purity and efficient.JE virus CJ50003 obtains like this: JE virus SA 14-14-2 (PDK) is adapted in the Vero tissue culture cells not being higher than under 35 ℃ the temperature, go down to posterity 4 times or more generations, select the virus of cultivation, according to the quantity of the transforming focus that in Vero and/or LLC-MK2 cell, forms, monitor the propagation of virus simultaneously.The virus that obtains from this adaptive method has 1 * 10 at least the culture supernatant of every milliliter of Cultivation of Vero 7The peak value titre of pfu, and reduced the incubation period of gathering in the crops.JE virus SA14-14-2 is a kind of virus strain of attenuation, its wild-type JE virus SA 14 adaptogen by making mosquito obtains for hamster kidney (PHK) tissue culture cells and PDK (PDK) tissue culture cells that (Kenneth H.Eckels waits the people, vaccine, 6513-518,1988).But PHK and PDK cell are not permitted by WHO, so they are unsuitable for being used for preparing vaccine for man.The Vero cell is subjected to the WHO permission to be used for human, so the JE virus strain CJ50003 that Vero adapts to is the good basis of producing vaccine for man.
Known SA 14-14-2 (PDK) virus belongs to flavivirus, and has following physico-chemical property: strand, and positive have an adopted rna gene group, and 5 ' methylated end is arranged, and 3 ' end does not have poly A structure.The size of rna gene group is about 11kb, and the nucleocapsid of genome and 13500Da (C) albumen is in assembled state.Virus also comprises in addition coating (E) albumen of film (M) albumen, 53000 Da of 8700Da and unstructuredness albumen NS1,2a, 2b, 3,4a, 5 etc.
The JE virus strain CJ50003 that Vero is adapted to goes down to posterity more than 30 generations in the Vero cell.By going down to posterity, virus titer and plaque morphology do not change, and this proposes virus and has stable phenotypic characteristic.
For the molecular basis of the biological property of understanding JE virus CJ50003, analyze the physico-chemical property of virus.Determine virus genomic base sequence with cDNA clone and sequencing.Found that three VITAMIN B4 of 1032,1506 and 1704 and 1769 s' guanine base is replaced by three guanines and a thymus pyrimidine in the JE CJ50003 virus respectively in the E protein gene of JE SA 14-14-2 (PDK).Therefore, 19 Threonine, 177 Threonine, 243 Methionin and 264 glutamine become L-Ala, L-Ala, L-glutamic acid and Histidine respectively in the proteic aminoacid sequence of E.As long as our research continues, when virus went down to posterity in the Vero cell, the proteic amino acid variation of E just remained unchanged.
When will the different virus injection of passage number is gone in the young mouse brain in the Vero cell, JE virus CJ50003 can not kill mouse.Therefore, we can say that the JE virus CJ50003 that adapts to Vero is a kind of attenuation and stable virus strain, it does not have or has only neurotoxicity seldom.This is that described virus is used for one of JE virus vaccines alive and/or key factor of inactivated vaccine.
The present invention also provide a kind of from the cell culture purified virus and need not freezing rough thing or during the method for purity material.Described method comprises the following steps: to remove cell debris, concentrating virus, and by sedimentation cell source material with carry out the super centrifugal purifying that comes of saccharose gradient, stepwise gradient, and measure viral fraction.More particularly, the invention provides a kind of method of producing purifying JE virus, this method comprises, in the existence of serum protein additive or not, in continuous cell line,,, and merge the virus-positive fraction with super centrifugal purification virus with the paramount titre of virus multiplication.
The described virus of propagation in the Vero tissue culture cells.Growing to the Vero cell that is paved with the CJ50003 virus infection in rolling bottle also cultivates.Available repeatedly harvesting method results virus.The the 2nd or the 3rd day (according to infection multiplicity) begins to gather in the crops culture supernatant after infection, adds fresh substratum in culture again.The culture of cultivating adding is again gathered in the crops culture supernatant after 2 days once more.Results can be after infection repeat 4 times in 8 or 9 days, and virus titer maintains and is higher than 10 7Pfu/ milliliter culture supernatant.Repeatedly harvesting method provides the viral yield that higher per unit is rolled bottle, thereby makes the present invention more adapt to the rule in market.In addition, this method also causes the CPE degree of infected cell to reduce.The CPE level that reduces is given in the JE vaccine of the present invention the extremely low-level residual cells composition of deriving.The culture supernatant of results can begin until purifying in 4 ℃ of preservations.The culture supernatant of results can realize clarificationization with common method known in the art, and these methods comprise, for example, and 1500g low-speed centrifugal 10 minutes, and/or be 0.45 membrane filtration by the aperture.The nutrient solution of 4 ℃ of preservation results is to concentrating.For virus is concentrated, nutrient solution is carried out ultrafiltration, collect retention then.In another concentration method, polyoxyethylene glycol (PEG) 8000 is dissolved in the nutrient solution to 10%, with throw out be dissolved in suitable damping fluid (phosphate buffered saline buffer for example, PBS, pH7.0) in.Carry out Protamine sulfates precipitation, removing DNA or from other material of cell, this can be by adding Protamine sulfates in the spissated viral solution and high speed centrifugation (for example 12000g) was realized in 5 minutes.For being further purified virus, density 15-60% continuously or the enterprising line density gradient of multistep saccharose gradient super centrifugal.The classification saccharose gradient is measured the virus in the fraction.The method that mensuration contains the virus-positive fraction comprises plaque test, hemagglutination test, polyacrylamide gel electrophoresis and antigen test (as immunity test).Low-level according to high virus titer and other impurity merges fraction so that further handle.By chromosomal DNA and the protein of test source, estimate the purity of the purified virus of merging in the Vero cell.The result shows, low respectively 2.5pg and the 2ng of reaching of the host cell DNA of per 5 microgram purifying JE viruses and Protein content, and this shows that above-mentioned purification process removed other impurity in the virus antigen effectively.Estimate that 1 liter of JE virus yield that infects nutrient solution is about 2.3 milligrams.
The present invention also provides a kind of deactivation JE virus to keep its method for enhancing antigenicity simultaneously to destroy its infectivity.Described method comprises, adds the formaldehyde of significant quantity, described virus and described reagent is cultivated under certain condition, thereby made described virus be inactivated.Specifically, with suitable damping fluid (for example PBS) fraction is merged thing and be diluted to suitable protein concentration, in the fraction merging thing of dilution, add formaldehyde.Carry out under 22 ℃ or 4 ℃ with the cultivation of formaldehyde.Under 22 ℃ or 4 ℃, completely destroy viral infection and do not lose virus antigenicity and need 4 days or 46 days at least respectively.Easy for large scale culturing should be chosen in the method for 22 ℃ of following deactivation JE viruses, and will cultivate time lengthening to 7 day, so that safe well-to-do amount to be provided.Yet effectively the example of inactivator is including, but not limited to formaldehyde.Usually, this can realize by chemistry or physics mode.Chemical ablation can be by handling and realize with (for example) enzyme, β propionic acid lactone, ethyleneimine or derivatives thereof and organic solvent (as Tween, Triton, Sodium desoxycholate and sulfo group sea pyridine (sulphohetain)).If desired, subsequently can in and the deactivation material; For example, can be neutralized with thiosulphate by the material of formalin-inactivated.Physical deactivation should be realized by high-energy radiation (as UV light, X-radiation or x radiation) by making virus.
But the JE vaccine can be made into the injection of liquor or suspensions.Stablizer such as carbohydrate (Sorbitol Powder, mannitol, starch, sucrose, dextran, glucose etc.), protein (albumin, casein etc.) be can add, protein reagent (bovine serum, skimmed milk etc.) and damping fluid (as alkali metal phosphate) contained.Prepared product can freeze-drying after adding stablizer, and its can vacuum or nitrogen preservation.If desired, can add one or more compounds with adjuvant effect.The suitable compound that is used for this purpose for example is aluminium hydroxide, aluminum phosphate or aluminum oxide, mineral oil (as Bayol, Marcol 52) and saponin.In addition, if desired, also can add one or more emulsifying agents in the viral material, for example Tween and span.
By measuring neutralizing antibody amount at virus, determine the effect of adjuvant, this neutralizing antibody is by giving also to be adsorbed in the vaccine non-activity virus generation of adjuvant.Effectively the adjuvant example includes, but are not limited to aluminium hydroxide.Carry out with the mice serum of described vaccine immunity that plaque reduces neutralization test (PRNT) and with neurotoxicity virus directtissima mice immunized, study the effectiveness of obtained vaccine.The result shows that described vaccine has the effectiveness of bringing out neutralizing antibody identical or better with comparable vaccine.
In order to study the possible variation of virus immunity originality that the different Vero of passage number adapts to, the vaccine that makes different passage numbers also compares the effectiveness of each vaccine.No matter continuous passage in the Vero cell does not have marked difference between its effectiveness of the vaccine that the virus of never simultaneous interpretation generation number makes.Therefore we can say that the JE virus strain CJ50003 that Vero adapts to has stable immunogenicity.
The following example has been described the attenuation JE virus of adaptation Vero cell of the present invention and the JE vaccine that comprises described attenuated virus of the present invention.Believe those skilled in the art according to aforementioned description and hereinafter embodiment implement the present invention fully.
Embodiment 1
The adaptation of SA 14-14-2 (PDK) virus in the Vero cell
With JE SA 14-14-2 (PDK), Madin-Darby canine kidney(cell line) (MDCK) is cultivated the 8th generation SA 14-14-2 virus, is enabled in the continuous passage in the Vero cell cultures.With the infection multiplicity inoculation Vero cell monolayer of JE SA 14-14-2 (PDK) with every cell 0.1pfu.At about 5%CO 2Air atmosphere under, not being higher than under about 35 ℃ temperature (about 32 ℃ to 35 ℃ usually, preferable about 35 ℃), make infected cell cultures at the 25cm that contains 5 milliliters of nutrition substratum 2Grow in the culturing bottle, this substratum is made up of the minimum essential medium of Eagle, has wherein added 10% foetal calf serum.Determine the existence of virus antigen by microscope observing cell pathology effect (CPE) and with various tests (as test of hemadsorption test (HA), plaque and enzyme linked immunosorbent assay (ELISA)), monitor viral growth.After infection the 5th day, when culture shows the virus titer peak value, results JE virus, and centrifugal clarification.Purifying list plaque and in the Vero cell, increasing from the clarificationization supernatant liquor.With the virus that increases vero cells infection again, further go down to posterity.Subsequently by aforesaid continuous virus infection, titration and plaque purification, carry out 30 generations of continuous passage.As shown in Figure 1, biography is after 4 generations in the Vero cell, and virus titer reaches about 4 * 10 7Pfu/ milliliter culture supernatant, and keep near this level in the going down to posterity afterwards.In addition, between the optimal period of virus results from the 2-3 that foreshortened to for the 4th generation in 5 days days of the first-generation.The obvious raising of virus yield is (from about 10 5Pfu/ml is to surpassing 10 7Pfu/ml) and the shortening of cultivating the time cause selecting in the Vero cell the 4th generation JE as the parent material that is used to prepare candidate JE vaccine.With the 4th generation JE in the Vero cell be designated as CJ50003 (Vero, PS4).Abbreviation PS refers to the viral passage number in designated cell.
Embodiment 2
The specificity analysis of CJ50003 virus; The order-checking of env gene and neurotoxicity research
In order to understand the molecular basis of CJ50003 strain biological nature, mensuration has 1500 nucleotide sequences of encoded packets membranin (E) gene of main neutralizing epitope, and and parental bacteria strain SA14-14-2 (PDK), SA 14-14-2 (PHK) and wild-type SA14 virus (Aihira, S. wait the people, Virus Genes, 5:95-109,1991; Ni, people such as H., JGen Virol.76:401-407,1995; Ni, people such as H., J Gen Virol.76:409-413,1995; Nitayaphan, people such as S., Virology 177:541-542,1990) sequence relatively.(Vero PS4) does the sequence analysis with CJ50003 virus.The result shows that (amino acid 280-500) is conservative fully for the C end regions, and N end regions (amino acid/11-279) shows sequence difference is arranged between the virus strain.Sudden change great majority in the N end regions are evenly distributed.The nucleotide sequence of the E protein gene of CJ50003 has 8 Nucleotide different (7 amino acid differences) with SA14/CDC, and SA14-14-2 (PDK) has 7 Nucleotide different (5 amino acid differences) with SA14/CDC.The result is summarized in the table 1.
The sequence of CJ50003 virus is different with the virus of disclosed SA 14-14-2 (PDK) in position, 5 place: 1032,1506,1704 and 1769 Nucleotide changes and causes SA 14-14-2 (PDK) and CJ50003 virus that 4 amino acid differences are arranged: 989 Nucleotide variation does not cause amino acid replacement.The CJ50003 virus of higher algebraically (i.e. 15 generations in generation to 30) does not disclose extra Nucleotide and changes.Therefore, there are 5 tangible Nucleotide variations and 4 tangible amino acid to change between CJ50003 and the parental virus SA 14-14-2 (PDK).These variations are stable when this virus goes down to posterity in cell cultures.243 Lys residue is to compare unique different residue with other attenuation JE virus among the SA 14-14-2 (PDK), and it is substituted by the Glu residue in CJ50003.
CJ50003 sequence and disclosed SA 14-14-2 (PHK) virus sequence (Aihira, people such as S., Virus Genes, 5:95-109,1991) are also different.1032 Nucleotide difference causes the amino acid difference of E19 position, but the variation of 989 Nucleotide does not cause amino acid replacement.1506 is identical with the Nucleotide of those positions among the SA14-14-2 (PHK) with 1704 Nucleotide in the CJ50003 virus, but different with the Nucleotide of those positions among the SA 14-14-2 (PDK).The N end regions alternative of CJ50003 and SA 14-14-2 (PHK) E gene is substantially the same, except the amino acid replacement at E19 place.
Compare with the sequence of parental generation SA 14 viruses, SA 14-14-2 (PHK), SA 14-14-2 (PDK) have 4 identical amino acid replacements with CJ50003 virus in E107, E138, E176 and E279 position.Wherein the amino acid of E138 and E176 position (Ni, people such as H., J Gen Virol.76:409-413,1995) (known these two amino acid have contribution to attenuation) is still conservative after Vero adapts to, and this prompting CJ50003 does not lose its attenuation feature.
Nucleotide between table 1 JE virus strain, SA14, SA 14-14-2 (PHK), SA 14-14-2 (PDK) and the CJ50003 and aminoacid sequence comparison position nucleotide amino acid NT AA SA14/ SA14/ SA14/ SA14-14-SA14-14-CJ50003 SA14/ SA14/ SA14/ SA14-14-SA14-14-CJ50003
USA CDC JAP 2/PHK 2/PDK USA CDC JAP 2/PHK 2/PDK989 E4 G G G U U G Leu Leu Leu Leu Leu Leu1032 E19 A A A A A G Thr Thr Thr Thr Thr Ala1052 E25 G A A A A A Leu Leu Leu Leu Leu Leu1061 E28 U U U C C C Asp Asp Asp Asp Asp Asp1217 E80 C C U U C C Ala Ala Ala Ala Ala Ala1296 E107 C C C U U U Leu Leu Leu Phe Phe Phe1389 E138 G G G A A A Glu Glu Glu Lys Lys Lys1503 E176 A A A G G G Ile Ile Ile Val Val Val1506 E177 A A A G A G Thr Thr Thr Ala Thr Ala1704 E243 G G G G A G Glu Glu Glu Glu Lys Glu1708 E244 G A A A A A Gly Gly Gly Gly Gly Gly1769 E264 G G G A G U Gln Gln Gln His Gln His1813 E279 A A A U U U Lys Lys Lys Met Met Met1921 E315 C U C U U U Ala Val Ala Val Val Val1977 E334 C C U C C C Pro Pro Ser Pro Pro Pro2293 E439 A G A G G G Lys Arg Arg Arg Arg Arg2441 E488 G A G A A A Gly Gly Gly Gly Gly Gly
AA: amino acid; NT: nucleotide position chain.
By being injected in the 4 week age BALB/c mouse brains (i.c.) the mouse neurotoxicity of test CJ50003 and parental generation SA 14-14-2 (PDK).The result is presented in the table 2.Compare with wild-type SA 14 viruses, SA 14-14-2 (PDK) and CJ50003 virus do not have marked difference for the lethality of young adult mice, and is all very low.Therefore, do not provide the neurovirulence phenotype at the bottom of the importing Vero cell based, and CJ50003 virus still has the attenuation characteristic for SA 14-14-2 (PDK).
Table 2. inoculated CJ50003 virus that Vero goes down to posterity 4 age in week mouse brain in toxicity.The passage number of PS representative in PDK or Vero cell.
Virus PFU inoculum size Log LD 50Ml -1LD 50/ PFU ratio
SA14(PDK，PS3) 2×10 7 6.5 0.17 *
SA14-14-2(PDK，PS8) 1.3×10 6 ＜1.5 b ＜0.00002 *
CJ50003(Vero，PS6) 3.4×10 7 ＜1.5 b ＜0.00001
CJ50003(Vero，PS15) 3.2×10 7 ＜1.5 b ＜0.00001
CJ50003(Vero，PS30) 3.6×10 7 ＜1.5 b ＜0.00001
People (Vaccine 6:513-518,1988) such as a:Kenneth H.Eckels.
B: after having inoculated undiluted virus, 10 none death of mouse.
The injection inoculation volume is 0.03 milliliter of every mouse in the brain.
Embodiment 3
Viral growth and purifying
Preparation seeding in the 5th generation virus [CJ50003 (Vero, PS5)] in the Vero cell, and freeze preservation deeply.The Vero cell is containing 10% foetal calf serum (FBS, Eagle minimum essential medium Gibco) (EMEM, Gibco) middle growth.With the roller bottle culture of seeding virus with the infection multiplicity vero cells infection individual layer of 0.01-0.1pfu/ cell.After 2 hours, wash culture 3 times in virus absorption, add the EMEM that does not contain serum with PBS, and 35 ℃ of cultivations.In the Vero cell cultures that infects, virus reached about 10 in 2 to 3 days after infection 7To 10 8The titre of pfu/ml.Although began in 2 or 3 days to the infection back to gather in the crops virus-4 altogether every 2 days in 8 or 9 days from infecting the back, virus titer still maintains and surpasses 10 7Pfu/ml, and CPE very a little less than.But after infecting 9 days, titre is lower than 10 7Pfu/ml (Fig. 2).The cutting of the centrifugal merging of 8000rpm 15 minutes makes supernatant liquor pass through 0.45 micron membrane filtration.With ultrafiltration (Ultrasette, Filtron, 100k) or PEG precipitation concentrating virus supernatant liquor.The centrifugal sedimentary virus of collection PEG method and be suspended from PBS or STE (10mM Tris pH7.2,1mM EDTA, 150mM NaCl) damping fluid in.Retention after the ultrafiltration is concentrated into 250 milliliters, with 100 milliliters of PBS sinks.After adding 0.5-2 mg/ml Protamine sulfates, make viral concentrated solution quenching on ice 2 hours, centrifugal 5 minutes of 10000rpm obtains supernatant liquor.By the super centrifugal spissated virus of purifying of coming on the saccharose gradient.Super centrifugal under 38000g, carrying out 18 hours.Make fraction electrophoresis (SDS-PAGE) on the polyacrylamide gel that contains the washing composition sodium lauryl sulphate.In SDS-PAGE, see nucleocapsid protein (C, 13500Da), membranin (M, 8700Da) and envelope protein (E, 53000Da) band (Fig. 3, group A).Detect envelope antigen (E) (Fig. 3, group C) with mouse anti JE viral monoclonal antibodies by the Western trace.Merge the virus-positive fraction (fraction 4 to 9) (Fig. 3, group B) that silver dyes other protein band that does not show in the gel except that viral protein, measure protein concentration with the Lowry method.Table 3 and table 4 have shown from the Vero that infects cultivates detailed results through two kinds of purification process (cross-flow ultrafiltration or PEG8000 precipitation).With the virus of the PBS of two volumes dilution purifying, add Tween80 to ultimate density be 0.01%, filter by 0.22 micron filter membrane.
Table 3. is by the concentrated and purified JE virus of cross-flow ultrafiltration
Population of samples amasss total pfu yield % total protein % yield, and (egg is than living
(milliliter) (pfu) (milligram) is white) (pfu/mg) merge culture supernatant 10,000 4.4 * 10 11100 600 100 7.3 * 10 7Filtering and concentrating liquid 200 4.0 * 10 1190 280 47 1.4 * 10 8Saccharose gradient merges thing 500 3.8 * 10 1186 42 7 9.0 * 10 90.22 micron filters 50 2.4 * 10 1155 23 3.8 1.0 * 10 10
Table 4. precipitates concentrated and purified JE virus by PEG8000
Population of samples amasss total pfu yield % total protein % yield, and (egg is than living
(milliliter) (pfu) (milligram) is white) (pfu/mg) merge culture supernatant 10,000 4.4 * 10 11100 600 100 7.3 * 10 7PEG precipitates 200 2.7 * 10 1161 40 6.7 6.8 * 10 9Saccharose gradient merges thing 500 2.5 * 10 1156 15 2.5 1.7 * 10 100.22 micron filters 50 1.6 * 10 1141 12 2 1.3 * 10 10
With measure the relative purity of (being pfu/ milligram albumen) more viral prepared product than living.From the virus that the concentrated solution purifying obtains roughly the same activity is arranged from the virus of concentrated solution purifying acquisition and by the PEG8000 precipitation by ultrafiltration.Also estimate the purity of the purified virus that merges in addition from the chromosomal DNA of Vero cell and albumen by test.The result shows, no matter is the sort of concentration method, low respectively 2.5pg and the 2ng of reaching of host cell DNA in per 5 microgram purifying JE viruses and protein content, and this proves that above-mentioned two kinds of purification process have all removed other impurity effectively from virus antigen.Yet aspect the albumen yield of purified virus, the purification process that adopts ultrafiltration is than adopting the good twice of the sedimentary purification process of PEG8000.
Embodiment 4
Inactivation of virus
The virus of purifying is used directly in the PBS dialysis back active attenuated vaccine of preparation or is used for the vaccine that formalin-inactivated prepares deactivation.Carry out at 22 ℃ or 4 ℃ with 0.018% formalin-inactivated.Fixed time interval is taken a sample, and directly measures infectious virus (Fig. 4) by plaque titration.Make sample blind passage generation (blind passage) on the Vero cell monolayer of in direct plaque test, being negative,, carry out plaque measurement then again so that increase low-level virus.Find that the complete inactivation infectivity needs 4 days in the time of 22 ℃, in the time of 4 ℃, need 46 days (table 5 and 6).During deactivation, by monitoring virus antigenicity with antigen Dot blot and polyclonal antiserum specimen.Show that by this experiment behind contact 0.018% formaldehyde 10 days (22 ℃) or 15 days (4 ℃), not detecting antigenicity has loss.
Carry out formalin-inactivated under following condition: 22 ℃ of at least 7 days or 4 ℃ at least 60 days have provided the well-to-do amount of security.After deactivation, add in 0.038% sodium metabisulfite and sample in free formaldehyde.Simultaneously with the PBS dialysis, then by 0.22 micron membrane filtration.
Table 5. at 4 ℃ with formalin-inactivated JE virus CJ50003
(HCHO) the JE virus (+HCHO) amplification of it JE virus
0 3.2×10 8 3.2×10 8 +
0.5 2.6×10 8 3.2×10 6 +
1 1.52×10 8 7.9×10 5 +
1.5 2.4×10 8 4.8×10 5 +
2 2.8×10 8 6.4×10 4 +
3 2.6×10 8 4.3×10 3 +
4 1.9×10 8 1300 +
5 2.4×10 8 285 +
6 2.8×10 8 480 +
7 1.5×10 8 200 +
11 1.5×10 8 0 +
22 1.4×10 8 0 +
32 1.2×10 8 0 +
46 1.0×10 8 0 -
60 1.0×10 8 0 -
Table 6 at 22 ℃ with formalin-inactivated JE virus
(HCHO) the JE virus (+HCHO) amplification of hour JE virus
0 3.2×10 8 3.2×10 8 +
3 3.0×10 8 1.3×10 6 +
6 2.7×10 8 1.1×10 5 +
12 2.5×10 8 3.5×10 5 +
24 1.2×10 8 140 +
36 1.2×10 8 0 +
48 1.2×10 8 0 +
72 1.1×10 8 0 +
96 1.1×10 8 0 -
360 1.0×10 8 0 -
Embodiment 5
The virus purifying of CJ50003, deactivation (PIV) and the immunogenicity of attenuated virus (LAV) in mouse of living
In mouse, use the immunogenicity of business-like Biken inactivated vaccine test LAV and PIV then.With three kinds of immunogens to one group 20 6 the week age BALB/c mouse do intraperitoneal (i.p.) immunity.With two kinds of inoculums, do not have adjuvant at interval 2 weeks to carry out immunity.For the second time two weeks after the immunity, obtain serum, merge and test with the PRNT method subsequently exist (table 7) of neutralizing antibody from each group mouse.As shown in table 7, accepting three kinds of NATs between immunogenic each group does not have significant difference.
Table 7. is induced the titre of neutralizing antibody immunogen dosage neutralizing antibody in PIV or LAV mice immunized a PIV 5 microgram 1: 320PIV 10 microgram 1: 320LAV 5 microgram 1: 320LAV 10 micrograms 1: 640Biken vaccine b1 dose 1: 320
A: the titre of neutralizing antibody is defined as the inverse of the serum dilution of the viral plaque minimizing 50% of the Nakayama that causes mouse brain to go down to posterity.
B: according to manufacturer's explanation, the Biken vaccine contains 5 microgram viral proteins (can precipitate with TCA) for 1 dose.
Further test the immunogenicity of PIV in mouse.With various dilution inactivation of viruses and or not with the inbreeding mouse that grows up of aluminium adjuvant immunity together.Be used in the salt solution or the salt solution of aluminium hydroxide (Rehydragel) in 500,50 and 5ng PIV subcutaneous immune one group 20 6 age in week BALB/c mouse.Mouse is accepted twice inoculation (being separated by for 3 weeks).In 3 weeks of back of immunity for the second time, merge the serum of respectively organizing mouse, go down to posterity the Nakayana virus strain as neutralization virus with mouse brain, the existing of test neutralizing antibody (table 8).PIV all is better than the Biken vaccine on all dosage, and adjuvant obviously make mouse to 50 and the immunne response of 500ng PIV improved about 4 and 8 times respectively.
Table 8. is with containing or the titre of neutralizing antibody is not relatively in the PIV mice immunized of aluminium hydroxide
The titre of immunogen dosage neutralizing antibody a
PIV 500ng 1∶160
PIV 50ng 1∶40
PIV 5ng 1∶20
PIV+ aluminium 500ng 1: 1280
PIV+ aluminium 50ng 1: 160
PIV+ aluminium 5ng 1: 20
1/10 dose of Biken vaccine 1: 80
1/100 dose of Biken vaccine 1: 10
1/1000 dose of Biken vaccine 1: 10
A: the titre of neutralizing antibody is defined as the inverse of the serum dilution of the viral plaque minimizing 50% of the Nakayama that causes mouse brain to go down to posterity.
B: according to manufacturer's explanation, the Biken vaccine contains 5 microgram viral proteins (can precipitate with TCA) for 1 dose.
The endogenous protective of test PIV is renderd a service in BALB/c mouse then.In order to carry out protection test, one group 10 3 age in week BALB/c mouse back leg subcutaneous vaccination salt solution or the deactivation JE virus in aluminium hydroxide (Rehydragel) salt solution.The contrast of age-matched is inoculated with PBS or aluminiferous heterogenetic antigen.Strengthen mouse with dose,equivalent after three weeks.In immunity 3 weeks of back, the 30 microlitre PBS that contain 500pfu mouse neurotoxicity JE virus (Nakayama adapts to mouse brain) by the encephalic inoculation attack mouse.The M ﹠ M to the of monitoring every day mouse under fire 21 days.As shown in table 9, show 90% protected through 50ng PIV mice immunized.In addition, through 50 and the mixture mice immunized of 5ng PIV and aluminium show 100% and 70% provide protection respectively, and 1/100 dose Biken vaccine has only been protected 50% the immune mouse that is subjected to.Compare, all mouse in the control group all began morbidity and dead in 5-7 days after attack.
Table 9 is subjected to vaccine mouse anti Nakayama virus aThe provide protection that immunogen is attacked
Immunogen dosage survival number
Contrast bN/A 0/10
PIV 500ng 10/10
PIV 50ng 9/10
PIV 5ng 3/10
PIV+ aluminium 500ng 10/10
PIV+ aluminium 50ng 10/10
PIV+ aluminium 5ng 7/10
The Biken vaccine c1/10 dose 10/10
1/100 dose 5/10 of Biken vaccine
1/1000 dose 3/10 of Biken vaccine
A: mouse is used 500pfu mouse neurotoxicity Nakayama virus attack then with 2 (being separated by for 3 weeks) immunity of test vaccine inoculation.
B: the contrast of age-matched is inoculated with PBS or aluminiferous heterogenetic antigen.
C: according to manufacturer's explanation, the Biken vaccine contains 5 microgram viral proteins (can precipitate with TCA) for 1 dose.
In order to study the immunology stability of CJ50003 virus when the Vero passage, independent purifying is the different virus of passage number in the Vero cell, estimates immunogenicity with the described method of table 8.As shown in table 10, between the vaccine that the virus of different virus passage number makes in the Vero cell, its ability that causes neutralizing antibody does not have marked difference, and this shows that CJ50003 virus is highly stable on Vero cell algebraically aspect the immunogenicity.
The effectiveness of the vaccine that table 10 makes with the JE virus of different virus passage number in the Vero cell
Immunogen aThe titre S.d. of dosage neutralizing antibody c
PIV-4ps 0.5 microgram 1: 150 20
PIV-6ps 0.5 microgram 1: 145 15
PIV-15ps 0.5 microgram 1: 130 28
PIV-20ps 0.5 microgram 1: 140 18
PIV-30ps 0.5 microgram 1: 160 13
A: immunogen (PIV-Xps); The vaccine of the deactivation of the purifying that makes from CJ50003 virus, the passage number of virus in the Vero cell is X.
B: the titre of neutralizing antibody is defined as the inverse of the serum dilution of the viral plaque minimizing 50% of the Nakayama that causes mouse brain to go down to posterity, and gets three separately mean values of result of experiment.50% end points is determined with Reed and Muench method.
C: standard deviation
Result shown in this paper shows that it all is promising adopting the deactivation JE virus vaccines of CJ50003 strain and attenuated vaccine alive.The present invention concentrates and is purified to the degree that is applicable to human body and their deactivation antigenicities are not had to measure loss for growth JE virus in the Vero cell, with them provide fastish, effective means.Find that these prepared products have immunogenicity and provide protection in the mouse body.

Claims (7)

1. the japanese encephalitis virus of an attenuation, it adapts to the Vero cell by uploading at the Vero cell to be commissioned to train to educate.
2. the japanese encephalitis virus of attenuation according to claim 1 is characterized in that, the plural number in the Vero cell is greater than 1 * 10 7The PFU/ milliliter is to the LD of young adult mice 50/ pfu is lower than 0.000001.
3. the japanese encephalitis virus of attenuation according to claim 1 and 2, wherein this virus is CJ50003.
4. Japanese encephalitis vaccine, it comprises the described japanese encephalitis virus of claim 1.
5. vaccine according to claim 4 wherein comes inactivation of viruses with inactivator.
6. vaccine according to claim 4, wherein virus is the japanese encephalitis virus of the attenuation of the work handled without inactivator.
7. according to claim 4,5 or 6 described vaccines, it also comprises pharmaceutically acceptable additive.
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